DESCRIPTION (Investigator's abstract): A wide variety of human malignancies are associated with aberrant transcription factors in a high proportion of cases. In none of these instances is that association stronger than that found between the EWS/FLI (and other EWS/ets) chimeric transcription factor and the Ewing Family Tumors, Ewing's Sarcoma and peripheral Primitive Neuroectodermal Tumor (PNET). Multiple lines of evidence demonstrate that the biology of these chimeric transcription factors is central to the development and sustenance of these tumors. The key to understanding the biology of these unique proteins is to elucidate those targets of biologic consequence which are aberrantly regulated as a result of EWS/FLI and its congeners. This proposal will accomplish this goal though the use of an expression cloning approach in which biologically relevant genes that act downstream of EWS/FLI will be isolated and identified. Using the MaRX expression cloning system and a model transformation system, genes mediating the in vitro phenotype of EWS/FLI will be elucidated. Genetic variables in the system will be tightly controlled. Measures will be taken to screen out false positives as well as to enhance the ability to detect important targets that may be expressed at low levels. Through a variety of measures, the role of these downstream effectors in human tumors will then be established. The in vivo phenotype of EWS/FLI will also be investigated. A population-based cycle cloning strategy will be used in an in vivo tumorigenesis assay. Those species that are enriched in this system will be characterized for their involvement in human tumorigenesis. Finally, an in vitro assay system will be used to isolate genes that work in concert with EWS/FLI to permit EWS/FLI transformability. Data from these studies will enhance our understanding of this enigmatic family of human tumors. This understanding will provide a valuable paradigm for the study of the wide array of malignancies that are associated with aberrant transcription factors.